Easy synergism for colistin-resistant KPC-producing Klebsiella pneumoniae: the E-test with supplemented agar

Abstract

FIG. 1. Minimum inhibitory concentrations of colistin on MuellerHinton agar alone (left) and supplemented with rifampin 32 mg/L (right) against Klebsiella pneumoniae carbapenemase (KPC)-producing The renewed interest in testing synergistic interactions of antimicrobial agents is mainly related to the challenge of antimicrobial resistance. Whereas time-kill curves and checkerboard methods represent the reference tests for screening synergy, the E-test with strips in a cross formation might be an interesting alternative, but requires that minimum inhibitory concentrations (MICs) of antibiotics have been previously determined [1]. However, agreement between the different tests seems to be quite variable [1]. To be clinically useful, a synergy test should be rapid and easily interpretable. We evaluated the synergistic activity of colistin (COL) plus rifampin (RIF) against 10 clinical isolates of COL-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) comparing the checkerboard method with the E-test on Mueller-Hinton agar, alone or supplemented with RIF. The 10 tested strains resulted in KPC3 production by polymerase chain reaction and Sequence Type (ST) 512 by multilocus sequence typing (MLST) analysis (http://www.pasteur.fr/recherche/ genopole/PF8/mlst/Kpneumoniae.html). COL MICs for the checkerboardmethodwere determinedwith brothmicrodilution in Mueller-Hinton broth alone and supplemented with RIF at the final concentration of 2, 4, 8, 16 and 32mg/L in 96-well microtiter plates. COL MICs also were determined with the E-test method on Mueller-Hinton agar alone or supplemented with RIF at the final concentration of 2, 4, 8, 16 and 32mg/L in Petri dishes (Fig. 1). According to the 2014 EUCAST Clinical Breakpoints, COL resistance was defined by MIC > 2 mg/L. With the checkerboard method, COL MICs for the 10 strains were 64 mg/L